It is currently not possible to distinguish the ductal carcinoma in situ (DCIS) that will develop into invasive breast cancer (IBC) from the DCIS that will not invade. As a result, many women are treated with potentially unnecessary interventions that are associated with short-term and long-term morbidities. Most attempts to define morphologic or molecular indicators of DCIS progression have focused on the DCIS epithelial cells. However, the stromal microenvironment surrounding DCIS is also altered, but the implications and origins of this stromal response have not been extensively studied. In the proposed project we will investigate a novel topic - the biomechanical forces exerted by DCIS, their consequences in the surrounding stroma, and the effect of the resulting stromal changes on the progression of DCIS to IBC. Our overall goals are two-fold - 1) to analyze the forces exerted by expanding ducts filled by DCIS on surrounding stroma and determine their impact on stromal fibroblast activation and the remodeling of the extracellular matrix (ECM), and the contributions of these forces to the growth and progression of DCIS and 2) to assess human DCIS tissues for the presence of the stromal changes induced by these forces. Ultimately, we intend to use the information gained to identify novel stromal biomarkers that will distinguish the aggressive DCIS that is likely to invade from the indolent form of DCIS and thereby prevent overtreatment. In Aim 1, novel three-dimensional, in vitro models of DCIS will be used to first measure the forces exerted by the intraductal accumulation of DCIS epithelial cells on the surrounding peri-ductal stroma, which will include the ECM and human breast fibroblasts. Then, quantitatively similar intraductal force, without the presence of DCIS epithelial cells, will be applied and the specific impact of these forces on stromal fibroblast activation will be assessed by gene expression profiling (RNAseq) and their impact on ECM remodeling and the resulting changes in the topography of the ECM scaffold will be determined by laser scanning microscopy. Finally, the effect of the force-induced changes, i.e., fibroblast gene expression and remodeled ECM topography, on the growth and invasion of DCIS epithelial cells will be measured. In Aim 2, the relevance of the force-induced changes to human DCIS will be assessed. DCIS tissues (formalin-fixed, paraffin-embedded) will be assessed for the presence of specific topographic features of the ECM around ducts involved by DCIS (by laser scanning microscopy) and for the expression of genes up-regulated or down-regulated by intraductal force (by immunohistochemistry). The presence of these changes will be compared in tissues with DCIS only and in those in which the DCIS has progressed to invasive breast cancer to begin to identify potential biomarkers that will predict the likelihood of DCIS progression to IBC.